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In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8,000 genes were detected with 7 × 10 4 unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions. Spatial analysis of RNAseq data is important. Here the authors report a method for transcriptome profiling combined with photo-isolation chemistry to allow determination of expression profiles specifically from photo-irradiated regions of interest which they use in mouse brains and embryonic tissues.

Epiblast stem cells (EpiSCs) are primed pluripotent stem cells and can be derived from postimplantation mouse embryos. We now show that the absence of canonical Wnt/β-catenin signaling is essential for maintenance of the undifferentiated state in mouse EpiSCs and in the epiblast of mouse embryos. Attenuation of Wnt signaling with the small-molecule inhibitor XAV939 or deletion of the β-catenin gene blocked spontaneous differentiation of EpiSCs toward mesoderm and enhanced the expression of pluripotency factor genes, allowing propagation of EpiSCs as a homogeneous population. EpiSCs were efficiently established and propagated from single epiblast cells in the presence of both XAV939 and the Rho kinase (ROCK) inhibitor Y27632. Cell transplantation revealed that EpiSCs were able to contribute to primordial germ cells and descendants of all three germ layers in a host embryo, suggesting that they maintained pluripotency, even after prolonged culture with XAV939. Such an improvement in the homogeneity of pluripotency achieved with the use of a Wnt inhibitor should prove advantageous for manipulation of primed pluripotent stem cells.

Mouse node cilia are posteriorly tilted to generate a leftward fluid flow and left/right asymmetry in the embryo, but how the tilt comes about was not known. The basal bodies of node cilia gradually shift from a central position towards the posterior side of node cells in a dishevelled and non-canonical Wnt signalling-dependent manner and follow a shift in Dvl localization to the posterior. Rotational movement of the node cilia generates a leftward fluid flow in the mouse embryo 1 because the cilia are posteriorly tilted 2 , 3 . However, it is not known how anterior-posterior information is translated into the posterior tilt of the node cilia. Here, we show that the basal body of node cilia is initially positioned centrally but then gradually shifts toward the posterior side of the node cells. Positioning of the basal body and unidirectional flow were found to be impaired in compound mutant mice lacking Dvl genes. Whereas the basal body was normally positioned in the node cells of Wnt3a −/− embryos, inhibition of Rac1, a component of the noncanonical Wnt signalling pathway, impaired the polarized localization of the basal body in wild-type embryos. Dvl2 and Dvl3 proteins were found to be localized to the apical side of the node cells, and their location was polarized to the posterior side of the cells before the posterior positioning of the basal body. These results suggest that posterior positioning of the basal body, which provides the posterior tilt to node cilia, is determined by planar polarization mediated by noncanonical Wnt signalling.

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5 + cardiac progenitor cells (CPCs) that express the Sry-type HMG box gene Sox17 from embryonic day (E) 7.5 to E8.5 specifically differentiate into the endocardium in mouse embryos. Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, likely as a consequence of down-regulation of NOTCH signaling. An unknown signal, regulated by Sox17 and required for nurturing of the myocardium, is responsible for the reduction in NOTCH-related genes in the mutant embryos. Our results thus provide insight into differentiation of the endocardium and its role in heart development.

Key Points Left–right (L/R) asymmetry provides a unique opportunity to study cellular and molecular mechanisms of asymmetry generation. Symmetry breaking in the mouse might involve unidirectional fluid flow around the node, which is generated by rotational movement of the monocilia. Transforming growth factor-β-related factors, Nodal and Lefty2, act as asymmetric signals. Nodal is a left-side determinant, whereas Lefty2 is an antagonist that restricts the duration and the site of Nodal action. Nodal and Lefty2 might comprise a reaction–diffusion system that amplifies L/R differences. The theoretical model describes two diffusible molecules, one of which is an activator that stimulates both its own synthesis and the synthesis of its partner, which is an inhibitor. The model requires that the inhibitor diffuses more rapidly than the activator. Midline structures are required to separate the left and the right halves of embryos. Lefty1 functions as a specific midline barrier. A transcription factor Pitx2 mediates Nodal signals and is responsible for generating the left-side morphology of many visceral organs. Although Nodal, the Lefty proteins and Pitx2 have conserved roles among vertebrates, diverse strategies might have been adopted for setting up asymmetric Nodal expression. The generation of morphological, such as left–right, asymmetry during development is an integral part of the establishment of a body plan. Until recently, the molecular basis of left–right asymmetry was a mystery, but studies indicate that Nodal and the Lefty proteins, transforming growth factor-β-related molecules, have a central role in generating asymmetric signals. Although the initial mechanism of symmetry breaking remains unknown, developmental biologists are beginning to analyse the pathway that leads to left–right asymmetry establishment and maintenance.

Congenital heart defects with heterotaxia are associated with pregestational diabetes mellitus. To provide insight into the mechanisms underlying such diabetes-related heart defects, we examined the effects of high-glucose concentrations on formation of the left–right axis in mouse embryos. Expression ofPitx2, which plays a key role in left–right asymmetric morphogenesis and cardiac development, was lost in the left lateral plate mesoderm of embryos of diabetic dams. Embryos exposed to high-glucose concentrations in culture also failed to expressNodalandPitx2in the left lateral plate mesoderm. The distribution of phosphorylated Smad2 revealed that Nodal activity in the node was attenuated, accounting for the failure of left–right axis formation. Consistent with this notion, Notch signal-dependent expression of Nodal-related genes in the node was also down-regulated in association with a reduced level of Notch signaling, suggesting that high-glucose concentrations impede Notch signaling and thereby hinder establishment of the left–right axis required for heart morphogenesis.

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a chemotactic G protein-coupled receptor expressed by leukocytes, such as granulocytes, macrophages, and activated T cells. Although there is growing evidence that BLT1 plays crucial roles in immune responses, its role in dendritic cells remains largely unknown. Here, we identified novel DC subsets defined by the expression of BLT1, namely, BLT1hi and BLT1lo DCs. We also found that BLT1hi and BLT1lo DCs differentially migrated toward LTB4 and CCL21, a lymph node-homing chemoattractant, respectively. By generating LTB4-producing enzyme LTA4H knockout mice and CD11c promoter-driven Cre recombinase-expressing BLT1 conditional knockout (BLT1 cKO) mice, we showed that the migration of BLT1hi DCs exacerbated allergic contact dermatitis. Comprehensive transcriptome analysis revealed that BLT1hi DCs preferentially induced Th1 differentiation by upregulating IL-12p35 expression, whereas BLT1lo DCs accelerated T cell proliferation by producing IL-2. Collectively, the data reveal an unexpected role for BLT1 as a novel DC subset marker and provide novel insights into the role of the LTB4-BLT1 axis in the spatiotemporal regulation of distinct DC subsets.

We have examined the role of Fam60a, a gene highly expressed in embryonic stem cells, in mouse development. Fam60a interacts with components of the Sin3a-Hdac transcriptional corepressor complex, and most Fam60a–/– embryos manifest hypoplasia of visceral organs and die in utero. Fam60a is recruited to the promoter regions of a subset of genes, with the expression of these genes being either up- or down-regulated in Fam60a–/– embryos. The DNA methylation level of the Fam60a target gene Adhfe1 is maintained at embryonic day (E) 7.5 but markedly reduced at E9.5 in Fam60a–/– embryos, suggesting that DNA demethylation is enhanced in the mutant. Examination of genome-wide DNA methylation identified several differentially methylated regions, which were preferentially hypomethylated, in Fam60a–/– embryos. Our data suggest that Fam60a is required for proper embryogenesis, at least in part as a result of its regulation of DNA methylation at specific gene promoters. As an embryo develops, its cells continue to divide and transform from unspecialized embryonic stem cells into the specialized cells that form the tissues and organs of the adult body. This complex process is controlled by a network of genes. Although most adult cells carry the same genes, different cell types each activate specific sets of genes, which ultimately gives them their unique properties. Likewise, developing cells also have unique patterns of gene expression that guide the cell’s development, behavior and its interaction with neighboring cells. For example, the gene Fam60a is highly active in embryonic stem cells, but until now, it was not known what role this gene had. To investigate this further, Nabeshima et al. studied mice that either had normal levels of Fam60a or reduced levels of Fam60a. The results showed that at a normal level, Fam60a was responsible for the intestines to develop properly. The guts of mice with reduced levels, however, grew very slowly. Moreover, Farm60a appears to regulate several other genes, and their activity was no longer controlled properly in these mice. Nabeshima et al. discovered that this was because Fam60a could interact with protein complexes responsible for repressing or activating genes. By changing the activity of these complexes, Fam60a could affect the activity of many other genes. A next step will be to find out how exactly Fam60a interacts with the protein complexes that affect the activity of genes. A better knowledge of how genes contribute to the development of an embryo may help understand the causes of miscarriage and find ways to prevent it.

Patterning of the mouse embryo along the anteroposterior axis during body plan development requires migration of the distal visceral endoderm (DVE) towards the future anterior side by a mechanism that has remained unknown. Here we show that Nodal signalling and the regionalization of its antagonists are required for normal migration of the DVE. Whereas Nodal signalling provides the driving force for DVE migration by stimulating the proliferation of visceral endoderm cells, the antagonists Lefty1 and Cerl determine the direction of migration by asymmetrically inhibiting Nodal activity on the future anterior side.

The determination of left-right body asymmetry in mouse embryos depends on the interplay of molecules in a highly sensitive structure, the node. Here, we show that the localization of Cerl2 protein does not correlate to its mRNA expression pattern, from 3-somite stage onwards. Instead, Cerl2 protein displays a nodal flow-dependent dynamic behavior that controls the activity of Nodal in the node, and the transmission of the laterality information to the left lateral plate mesoderm (LPM). Our results indicate that Cerl2 initially localizes and prevents the activation of Nodal genetic circuitry on the right side of the embryo, and later its right-to-left translocation shutdowns Nodal activity in the node. The consequent prolonged Nodal activity in the node by the absence of Cerl2 affects local Nodal expression and prolongs its expression in the LPM. Simultaneous genetic removal of both Nodal node inhibitors, Cerl2 and Lefty1, sustains even longer and bilateral this LPM expression.